# Quantitative Proteomics and CRISPR/Cas9 Editing Reveal UPR‐Mediated Control of Immunoglobulin Homeostasis in Hybridomas

## TL;DR

This study shows how the unfolded protein response (UPR) controls antibody production in hybridoma cells and introduces a new fluorescent system to monitor this process in real time.

## Contribution

A novel UPR-based fluorescent reporter system was developed for real-time monitoring of antibody stability in hybridomas.

## Key findings

- The UPR pathway regulates aberrant immunoglobulin chain production in hybridomas.
- A strong negative correlation (R2 = 0.86) was found between UPR activation and IgG levels.
- Fluorescence-activated cell sorting identified dysfunctional subclones with impaired Ig secretion.

## Abstract

Despite their tremendous economic value, monoclonal antibodies are often compromised by the loss of immunoglobulin (Ig) chains, which disrupts antibody homeostasis and quality control. Through subclone screening and characterization, we identified that the loss of Ig production impaired the recognition ability of hapten‐specific hybridomas. Proteomic analysis further highlighted the critical role of the unfolded protein response (UPR) pathway in regulating aberrant Ig chain production. Using CRISPR/Cas9‐mediated knockout and rescue experiments, we revealed the importance of the UPR pathway in facilitating hybridoma antibody production by targeting Xbp1s, an active transcription factor downstream of UPR signaling. With CRISPR/HDR, we inserted a fluorescent mGFP tag into the endogenous Hspa5 gene (encoding BiP, the master regulator of the UPR pathway), enabling in situ and real‐time monitoring of UPR activation. A strong negative correlation (R2 = 0.86) was observed between intracellular mGFP signals and IgG levels in the engineered system, indicating a close relationship between UPR activation and Ig production. Fluorescence‐activated cell sorting of high‐mGFP populations identified two dysfunctional subclones that failed to secret Ig, validating the system’s effectiveness in tracing Ig homeostasis. In summary, this study provides new insights into UPR‐mediated regulation of Ig synthesis and offers a novel UPR‐based reporter system for monitoring antibody stability.

BCR sequencing and subclone analysis correlated immunoglobulin (Ig) chain loss in dysfunctional hybridomas with disrupted monoclonal antibody homeostasis. Proteomics‐guided CRISPR/Cas9 editing revealed that the unfolded protein response (UPR) regulates aberrant Ig synthesis. A novel UPR‐based fluorescent reporter system was further established via CRISPR/HDR, enabling real‐time monitoring of UPR activation and antibody stability in hybridomas.